Identification of novel miRNAs and miRNA expression profiling in embryogenic tissues of <i>Picea balfouriana</i> treated by 6-benzylaminopurine

<div><p>Here, we compared miRNA expression profiles in embryonic cell cultures of the conifer <i>Picea balfouriana</i> following application of the synthetic cytokinin 6-benzylaminopurine (6-BAP). We used next-generation sequencing to analyze three libraries of small RNAs from the treated embryogenic cell cultures and generated 24,000,000 raw reads from each of the libraries. Over 70 differentially regulated micro RNA (miRNA) families (≥2 fold change in expression) were identified between pairs of treatments. A quantitative analysis showed that miR3633 and miR1026 were upregulated in tissues with the highest embryogenic ability. These two miRNAs were predicted to target genes encoding receptor-like protein kinase and GAMYB transcription factors, respectively. In one library, miR1160, miR5638, miR1315, and miR5225 were downregulated. These four miRNAs were predicted to target genes encoding APETALA2, calmodulin-binding protein, and calcium-dependent protein kinase transcription factors. The expression patterns of the miRNAs and their targets were negatively correlated. Approximately 181 potentially novel <i>P</i>. <i>balfouriana</i> miRNAs were predicted from the three libraries, and seven were validated during the quantitative analysis. This study is the first report of differential miRNA regulation in tissues treated with 6-BAP during somatic embryogenesis. The differentially expressed miRNAs will be of value for investigating the mechanisms of embryogenic processes that are responsive to 6-BAP in <i>P</i>. <i>balfouriana</i>.</p></div

Activities of antioxidant enzymes in 6-BAP-treated tissues in proliferation and maturation stages.

<p><b>(a)</b> Activity of superoxide dismutase (SOD). <b>(b)</b> Activity of peroxidase (POD). Tissues in proliferation and maturation stages were collected after being transferred to new media for 7 d. Lowercase letters (a, b, c) indicate significant differences (<i>p</i> ≤ 0.05).</p

Expression stability and ranking of reference genes as calculated by geNorm.

<p>Expression stability and ranking of reference genes as calculated by geNorm.</p

Levels of plant hormones in 6-BAP-treated tissues in proliferation and maturation stages.

<p><b>(a)</b> Levels of 3-acetic acid (IAA). <b>(b)</b> Levels of zeatin-riboside (ZR). <b>(c)</b> Levels of gibberellic acid (GA₃). <b>(d)</b> Levels of abscisic acid (ABA). Tissues in proliferation and maturation stages were collected after being transferred to new media for 7 d. Lowercase letters (a, b, c) indicate significant differences (<i>p</i> ≤ 0.05).</p

Stability assessment of the candidate reference genes by NormFinder.

<p>Stability assessment of the candidate reference genes by NormFinder.</p

Numbers of differentially expressed miRNAs and their targets among the three 6-BAP-treated libraries.

<p>Numbers of differentially expressed miRNAs and their targets among the three 6-BAP-treated libraries.</p

Orthologs of putative novel miRNAs conserved in other species.

<p>Orthologs of putative novel miRNAs conserved in other species.</p

Validation of differentially regulated miRNAs and their targets by qRT-PCR.

<p>(a)The x-axis shows the miRNAs validated in this study. The y-axis shows the log₂ ratio of their expression in the 3.6 μM 6-BAP versus the 5.0 μM 6-BAP libraries. Three biologically independent replicates were analyzed for each qRT-PCR; <b>(b)</b>The x-axis shows the miRNAs validated in this study. The y-axis shows the log₂ ratio of their expression in the 3.6 μM 6-BAP versus the 2.5 μM 6-BAP libraries. Three biologically independent replicates were analyzed for each qRT-PCR.</p

GO analysis and KEGG pathway enrichment of target genes of known miRNAs.

<p>GO analysis and KEGG pathway enrichment of target genes of known miRNAs.</p

Stability assessment of the candidate reference genes by BestKeeper.

<p>Stability assessment of the candidate reference genes by BestKeeper.</p